Canadian Journal of Microbiology

Isolation and characterization of a bacterium able to degrade high concentration of iprodione

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2017-0185.R1

Manuscript Type: Article

Date Submitted by the Author: 30-Sep-2017

Complete List of Authors: Cao, Li; Hexi University Shi, Wenhong; Hexi University Shu, Rundong; Hexi University Pang, Jian;Draft Hexi University Liu, Yuetao; Hexi University Zhang, Xiaohua; Hexi University Lei, Yuming; Hexi University, College of Agriculture and Biotechnology

Is the invited manuscript for consideration in a Special N/A Issue? :

Keyword: Biodegradation, Iprodione, Metabolite, Microbacterium

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Isolation and characterization of a bacterium able to degrade high concentration of

iprodione

Li Cao, Wenhong Shi, Rundong Shu, Jian Pang, Yuetao Liu, Xiaohua Zhang, Yuming Lei *

College of Agriculture and Biotechnology , Hexi University, Zhangye , Gansu , 734000, China

Draft

*Corresponding author. Tel.: +869368281523.

Email address: [email protected]

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Abstract: A bacterial strain CQH1 capable of mineralizing iprodione was isolated and characterized. In combination with morphological, physiological & biochemical characters, and the phylogenetic analysis of 16S rRNA gene sequence, strain CQH1 was identified as

Microbacterium sp.. It could use iprodione and 3,5dichloroaniline as the sole carbon source and energy source for growth. It completely degraded 100 mg L 1 of iprodione within 96 h at

30 °C. During the degradation of iprodione by strain CQH1, two compounds were detected in GCMS analysis and recognized as N(3,5dichlorophenyl)2,4dioxoimidazolidine and

3,5dichloroaniline, respectively. So, the biodegradation pathway of iprodione by strain

CQH1 was proposed. This is the first report of iprodionemineralizing strain from the genus of Microbacterium , and strain CQH1 might be a promising candidate for its application in the bioremediation of iprodionecontaminated environments.

Draft

Key words: Biodegradation; Iprodione; Metabolite; Microbacterium

1. Introduction

Iprodione [3(3,5dichlorophenyl)Nisopropyl2,4dioxoimidazolidine1carboxamide] is a dicarboxamide fungicide that inhibits DNA and RNA synthesis, cell division and cellular metabolism in fungi (Davidse. 1986), which is commonly employed in a variety of greenhouse and field crops to control fungal infestations by Botrytis cinerea , Alternaria sp.,

Monilinia fructigena , Rhizoctonia solani , Sclerotinia sclerotiorum , Aspergillus sp.,

Penicillium sp., Sclerotinia sp. and other fungal pathogens (Miñambres et al. 2010; Wang et al.

2012; Morales et al. 2013; Grabke et al. 2014; Campos et al. 2015). Interestingly, benzimidazole resistance is now widespread because of its frequent and indiscriminant use, while iprodione was developed in response to resistance problems of benzimidazoles

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(Sequinatto et al. 2013).

Iprodione is moderately persistent in soil with a halflife time of 7–60 d depending on the

environmental conditions (Carmona et al. 2001). It is relatively mobile in the soil environment

and leachable into groundwater and surface water, so its residues have been detected in many

environment samples (Derbalah et al. 2003; Omirou et al. 2009; Wang et al. 2012; Sequinatto

et al. 2013; Allen et al. 2015). Iprodione has been classified as a probable carcinogen, because

iprodione shows relatively high toxicity to crustaceans, moderately toxicity to fish and slight

toxicity to birds (Morales et al. 2013). It could delay male rat pubertal development, reduce

serum testosterone levels, decrease ex vivo testicular testosterone production, and even have

potential oncogenic effect (Washington and Tchounwou, 2004; Blystone et al. 2007). There

are also a few reports that iprodione may inhibit the microbes of environment and have an

impact on the diversity of soil microbialDraft communities (Wang et al. 2004; Verdenelli et al.

2012). So, the presence of iprodione residues is a matter of serious concern.

Microbial biodegradation is the main mechanism responsible for the dissipation of

iprodione. To date, several bacterial strains capable of degrading iprodione have been reported,

including sp. strain MA6 (Athiel et al. 1995), four combined Pseudomonas

strains (Mercadier et al. 1997) and Arthrobacter sp. strain C1 (Campos et al. 2015). During

the degradation of iprodione, N(3,5dichlorophenyl)2,4dioxoimidazolidine,

3,5dichlorophenylurea acetate and 3,5dichloroaniline were the most frequently detected

intermediate metabolites (Athiel et al. 1995; Mercadier et al. 1997; Campos et al. 2017), and

then the biodegradation pathway of iprodione was proposed. Bioremediation has received

increasing attention as a reliable, effective, safe, costeffective and promising approach to

clean up polluted environments (Rajendran et al. 2003; Chen et al. 2011; Cao et al. 2013).

Several pesticides including parathion, atrazine, and chlorpyrifos have been successfully

removed from soil and aquatic environments using microbes (Munnecke and Hsieh 1974;

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Singh and Walker 2006; Yadav 2014; Zhang 2015). However, no report has been made on bioremediation of iprodione. The objective of the present study was to isolate a bacterium able to degrade high concentration of iprodione, to study the effects of pH and temperature on biodegradation of iprodione, to analyze the metabolites appeared during the degradation of iprodione, and finally to provide a candidate for the bioremediation of iprodione contaminated environments.

2. Materials and methods

2.1. Chemicals and media

Iprodione and 3,5dichloroaniline (analytical standards: >97% purity) were purchased from SigmaAldrich of USA. DNA polymerase, T4 ligase, DNA gel extraction kit, plasmid extraction kit for DNA manipulationDraft were purchased from Takara Biotechnology Co. Ltd.

(Dalian, China). Other conventional reagents used in this study were of the highest analyticalreagent grade.

1 The mineral salts medium (MSM) contains (g L ): K 2HPO 4 1.5, KH 2PO 4 0.5, MgCl 2 0.2,

NaCl 1.0, NH 4NO 3 1.0 , pH 7.0. Iprodione or 3,5dichloroaniline was used as the sole carbon source. Concentrated stock solution of iprodione or 3,5dichloroaniline (10 g L 1) was prepared in acetone solution, which was sterilized via membrane filtration and diluted into sterile flask to achieve the desired concentrations, then the corresponding volume of medium was poured into the flasks after acetone has volatilized. Luria–Bertani (LB) medium contains (g L 1): tryptone 10.0, NaCl 10.0 and yeast extract 5.0, pH 7.0. Solid medium were prepared by adding 15 g L 1 agar into above liquid media.

2.2. Isolation and identification of iprodionedegrading

Iprodionedegrading bacteria were isolated via the enrichment culture technique. The samples were collected from the soil of corn field located in the south of Zhangye in Gansu

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province of China (38°3216′′ N and 99°25′38′′ E) with previous exposure to iprodione. The 10

g of sample was added into a 250 mL flask with 100 mL of sterile MSM containing 100 mg

L1 iprodione and incubated on a rotary shaker (160 rpm) at 30 °C for 7 days. The suspension

(5 mL) was transferred to fresh MSM containing 100 mg L 1 iprodione and incubated for

another 7 days. After five rounds of enrichment, the culture was diluted and spread onto solid

MSM plates containing 100 mg L 1 iprodione and incubated at 30 °C for 5 days.

Iprodionedegrading ability was observed in terms of the formation of transparent halos

surrounding the colonies. A bacterial strain designated as CQH1 that formed transparent

halos around their colonies was selected for further study.

Strain CQH1 was identified according to Bergey’s Manual of Determinative

Bacteriology (Holt et al. 1994). Its 16S rRNA gene was amplified via polymerase chain

reaction (PCR) using standard proceduresDraft (Lane, 1991). The PCR product was purified,

ligated into pMD19T, and transformed into E. coli DH5α. An automatic sequencer was used

to determine the 16S rRNA gene sequence. The 16S rRNA gene sequence was deposited at

GenBank (Accession No. KX618653). Multiple alignment analysis of different 16S rRNA

gene sequences in the GenBank database was performed using ClustalW 1.8.3 with default

settings. The sequence alignments were analyzed using MEGA5 software. The distances were

calculated using the Kimura twoparameter distance model. Unrooted phylogenetic trees were

constructed using the neighborjoining method. Datasets were bootstrapped 1000 times

(Saitou and Nei, 1987).

2.3. Biodegradation of iprodione in liquidculture condition

Strains were collected by centrifugation at 6000×g for 5 min at room temperature after

precultured for 16 h in LB medium at 30 °C on a shaker at 160 rpm. The cell pellets were

washed twice with MSM and adjusted to an optical density of 1.0 at 600 nm. Unless

otherwise stated, the cells were inoculated at 3% (v/v) into a 250 mL flask containing 100 mL

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of MSM at pH 7.0 amended with 100 mg L1 iprodione or 30 mg L 1 3,5dichloroaniline as sole carbon and energy sources and incubated at 30 °C and 160 rpm in a rotary shaker. At each sampling point, the samples were used to determine the concentration of iprodione, the concentration of 3,5dichloroaniline, and cell density (OD 600 nm). Uninoculated medium was used as the control. All experiments were replicated for three times.

2.4. Effect of pH and temperature on biodegradation of iprodione

The MSM at different pH (4.08.0) containing iprodione (100 mg L 1) were used to study the effect of pH on degradation rate of iprodione at 30 °C. The degradation rate of iprodione at pH 7.0 was also determined in MSM containing iprodione (100 mg L 1) at different temperature conditions (20 °C40 °C). The cells of strain CQH1 were inoculated at a 3% (v/v) level and incubated at 160 rpm in a rotary shaker. Uninoculated medium was used as the control. All experiments were replicatedDraft three times. The rate of degradation was calculated as follows: [Initial concentration of iprodioneconcentration of iprodione after degradationabiotic control (uninoculated medium)]*100%/ [Initial concentration of iprodione

abiotic control (uninoculated medium)].

2.5. Analytical methods

Samples were centrifuged at 12,000 ×g for 5 min. The supernatant was then collected and extracted twice with an equal volume of dichloromethane. The mixture was vortexed for

2 min. After centrifugation at 12,000 rpm for 5 min at room temperature, the dichloromethane layer was recovered and dried over anhydrous sodium sulfate. The organic phase was evaporated at room temperature. The residues were redissolved in 0.5 mL of ethanol and filtered through a 0.2 mpore filter. An aliquot was used to detect iprodione and

3,5dichloroaniline by reversephase HPLC (600 Controller, Rheodyne 7725i Manual injector and 2487 Dual λ Absorbance Detector; Waters Co., Milford, MA) with a mobile phase of methanol: water (80:20, v:v). The separation column for the HPLC (4.6 mm × 250 mm × 5

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µm) was filled with Kromasil 1005C18. The flow rate was 0.8 mL min 1 and the injection

volume was 20 L. The retention time for iprodione and 3,5DCA were 8.6 min and 4.2 min,

respectively.

Another aliquot was used to detect the metabolites of iprodione by GC/MS. The GC/MS

was performed on a Shimadzu GC14B equipped with an electron capture 63 Ni detector (ECD,

Shimadzu, Kyoto, Japan) and an OV225 capillary GC column (30 m × 0.25 mm × 0.25 m;

OV). The temperatures of the column, injector, and detector were maintained at 210 °C,

250 °C, and 280 °C, respectively. The carrier gas was nitrogen, with a flow rate of 40 mL

min 1.

3. Results

3.1. Isolation and characterization of strain CQH1

Strain CQH1 was isolated by usingDraft the enrichment method. It was a coryneform rods ，

nonsporeforming, grampositive bacterium. It was positive for catalase, gelatin, and casein

hydrolyses test, but negative for the oxidase test. After 30 hours of incubation on LB plates,

colonies of strain CQH1 were bright yellow, circular with entire edge and smooth surface. Its

16S rRNA gene fragment (GenBank Accession No. KX618653) was 1356 bp and compared

with sequences in the GenBank. It showed high similarity with the strain from the genus of

Microbacterium , and the highest similarity of 99.8% was with strain Microbacterium

paraoxydans CF36 (GenBank Accession No. AJ491806). A phylogenetic tree based on known

representatives of the describing Microbacterium species and other species is presented in Fig.

1. Based on the above characteristics, strain CQH1 was identified as Microbacterium sp.

CQH1.

[Fig. 1 here]

3.2. Degradation of iprodione by strain CQH1

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The kinetics of degradation of iprodione and 3,5dichloroaniline, and growth of strain

CQH1 were investigated simultaneously in MSM containing 100 mg L 1 iprodione or 30 mg

L1 3,5dichloroaniline (Fig. 2). Bacterial growth was accompanied by the degradation of iprodione and the production of 3,5dichloroaniline. Iprodione decreased quickly to 41.2 mg

L1 in the first 16 h and then completely disappeared within 96 h of cultivation accompanied by the accumulation of 3,5dichloroaniline. Only approximate 39% iprodione disappeared in the noninoculated samples within 128 h at pH 7.0 and 30 °C. The accumulation

3,5dichloroaniline reached a maximum after 64 h and then was not detected after 128 h (Fig.

2A). Strain CQH1 could also use 3,5dichloroaniline as sole carbon and energy sources to grow and completely degrade 3,5dichloroaniline of 30 mg L 1 within 120 h at pH 7.0 and 30 °C (Fig. 2B), which suggested thatDraft iprodione could be mineralized by strain CQH1. Cell density (OD 600 nm ) gradually increased with the degradation of iprodione or

3,5dichloroaniline, respectively reached the maximum level after 96 h and 72 h (Fig. 2), which revealed that both of iprodione and 3,5dichloroaniline were used as the sole source of carbon and energy for the growth of strain CQH1.

[Fig. 2 here]

3.3. Effect of temperature on iprodione biodegradation

Because temperature is an important factor that influences the degradation of xenobiotic compounds by microorganisms, we investigated the effect of temperature on degradation of iprodione by strain CQH1 (Fig. 3). Taking into account of the iprodione degradation under abiotic control (Fig. 2), the rate of degradation was calculated with the formula mentioned in materials and methods section. Strain CQH1 was able to degrade iprodione at temperatures ranging from 20 °C to 40 °C. The rate of iprodione degradation gradually increased,

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reached the maximum level of 88.6% after 64 h at 35 °C (Fig. 3).

[Fig. 3 here]

3.4. Effect of initial pH on biodegradation of iprodione

Initial pH was also an important factor that influenced the degradation of xenobiotic

compounds by microorganisms, so the capacity of strain CQH1 to degrade iprodione in a

range of pH was investigated. Strain CQH1 was capable of degrading iprodione in a wide pH

range from 4 to 8 (Fig. 4). The degradation rate of iprodione reached the maximum level of

84.8% at pH 7. Though iprodione was vulnerable to hydrolysis at alkaline pH (Szeto et al.

1989; Campos et al. 2015), but higher pH value above 7.0 did not enhance the biotic

degradation rate of iprodione of strainDraft CQH1.

[Fig. 4 here]

3.5. Analysis of iprodione biodegradation metabolites by strain CQH1

During the degradation of iprodione by strain CQH1, three compounds A, B and C with

retention time of 8.6, 7.2 and 4.2 min were detected in the GC analysis, respectively (Fig. 5),

and they were further analyzed using GC/MS. The positiveion chemical ionization of the

product in standard GC/MS showed several prominent protonated molecular ions. In standard

GC/MS, compound A has prominent protonated molecular ions at m/z 147

+ + [MCO 2NH 2CH(CH 3)22HCl3H] , m/z 167 [M2HClCONHCH(CH 3)2] , m/z 279

[MCHCl] + and m/z 329 [M] +; compound C has prominent protonated molecular ions at m/z

+ + + 71 [M2HClNH 2] , m/z 85 [M2HCl] and m/z 161 [M] (Fig. 5). The compounds A and C

were elucidated as the same as that of authentic iprodione and 3,5dichloroaniline,

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respectively. The prominent protonated molecular ions at m/z 187 [MCCOOH] +, m/z 244

+ + + [M] , m/z 124 [MCO 2NHCH 2COOH2H] and m/z 161 [MHClCOOH] indicate that compound B is N(3,5dichlorophenyl)2,4dioxoimidazolidine. So, the compounds A, B and

C were recognized as iprodione, N(3,5dichlorophenyl)2,4dioxoimidazolidine and

3,5dichloroaniline, respectively. Strangely, 3,5dichlorophenylurea acetate was not detected in our study. But it was frequently detected in the degradation of iprodione by other iprodionedegrading bacterial strains (Athiel et al. 1995; Mercadier et al. 1997; Campos et al.

2017). In these strains, N(3,5dichlorophenyl)2,4dioxoimidazolidine was metabolized to

3,5dichloroaniline via 3,5dichlorophenylurea acetate. Based on these results and the growth of strain CQH1 on iprodione and 3,5dichloroaniline (Fig.1), we propose that strain CQH1 can hydrolyze iprodione to N(3,5dichlorophenyl)2,4dioxoimidazolidine, further metabolize to 3,5dichloroaniline, andDraft finally completely mineralize 3,5dichloroaniline.

Therefore, the proposed degradation pathway of iprodione by strain CQH1 was shown in Fig.

6. Strain CQH1 might have similar metabolic pathway of transforming iprodione into

3,5dichloroaniline with Arthrobacter sp. strain MA6 (Mercadier et al. 1997), but strain

CQH1 can further degrade 3,5dichloroaniline, and completely mineralize iprodione.

[Fig. 5 here]

[Fig. 6 here]

4. Discussion

A few bacterial strains capable of degrading iprodione have been isolated and characterized. The first pure bacterial culture Arthrobacter sp. strain MA6 able to metabolize iprodione was isolated in 1995, and the iprodione concentration in sterile mineral medium at pH 6.5 decreased from 30 mmol L 1 to approximately 4 mmol L 1 after 7 days (Athiel et al.

1995). The combination of Pseudomonas fluorescens , Pseudomonas sp. and Pseudomonas

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paucimobilis could degrade approximately 80 µmol L 1 of iprodione after 180 h (Mercadier et

al. 1997). Arthrobacter sp. strain C1 was able to degrade iprodione with only slight formation

of 3,5DCA, and the presence of atrazine, chlorpyrifos and isoproturon did not significantly

affected its degradation capacity against iprodione (Campos et al. 2015) . In this study, an

iprodionemineralizing strain was isolated. It showed the highest similarity of 99.8% with

strain Microbacterium paraoxydans CF36. It was identified as Microbacterium sp. CQH1

based on the morphological, physiological & biochemical characters, and the phylogenetic

analysis of 16S rRNA gene sequence. It completely degraded 100 mg L 1 of iprodione within

96 h at pH 7.0 at 30 °C and mineralized iprodione. To the best of our knowledge, this is the

first report of iprodionemineralizing strain from the genus of Microbacterium , which

enriches the diversity of iprodione degraders in the environments and provides a promising

candidate for the bioremediation of iprodioneDraft contaminated environments.

Strain MA6 converted iprodione into N(3,5dichlorophenyl)2,4dioxoimidazolidine

and (3,5dichlorophenylurea) acetic acid, which was further transformed into

3,5dichloroaniline by other soil microorganisms (Mercadier et al. 1996). Strains

Pseudomonas fluorescens and Pseudomonas sp. could only transformed iprodione into

N(3,5dichlorophenyl)2,4dioxoimidazolidine and (3,5dichlorophenylurea) acetic acid, and

strain Pseudomonas paucimobili transformed N(3,5dichlorophenyl)2,4dioxoimidazolidine

or (3,5dichlorophenylurea) acetic acid into 3,5dichloroaniline (Mercadier et al. 1997).

Arthrobacter sp. strain C1 is able to hydrolyze iprodione to intermediate metabolites which

are then further metabolized to 3,5dichloroaniline and glycine (Campos et al. 2015; Campos

et al. 2017). Both strain YJNG and strain CQH1 were isolated by the enrichment culture

technique, and identified as the genus of Microbacterium (Yang et al. 2017), but the similarity

of their 16S rRNA sequence is about 99.1%. Strain YJNG could only hydrolyze iprodione to

N(3,5dichlorophenyl)2,4dioxoimidazolidine (Yang et al. 2017), whereas strain CQH1

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could mineralize iprodione. Strain CQH1 showed a high degradation rate of iprodione in a wide pH and temperature range. Suitable pH and temperature significantly contribute to the faster degradation of iprodione by strain CQH1. It might be one of effective iprodione degraders isolated. However, only a few data concerning the degradation metabolites of iprodione are obtained in this study. Further studies are needed to elucidate the degradation pathway of iprodione and its molecular mechanism.

Acknowledgments ：：：This work was supported by Chinese National Natural Science Fund

(31560029), Key Laboratory of Hexi Corridor Resourses Utilization Fund (XZ1401), Gansu

Agriculture Science and Technology Innovation Fund (GNCX201310). Draft References

Allen, G., Halsall, C.J., Ukpebor, J., Paul, N.D., Ridall, G., Wargent, J.J., 2015. Increased

occurrence of pesticide residues on crops grown in protected environments compared to

crops grown in open field conditions. Chemosphere 119, 14281435.

Athiel, P., Mercadier, C., Vega, D., Bastide, J., Davet, P., Brunel, B., CleyetMarel, J.C., 1995.

Degradation of iprodione by a soil Arthrobacter like strain. Applied and Environmental

Microbiology 61 (9), 32163220.

Blystone, C.R., Lambright, C.S., Furr, J., Wilso, V.S., Gray, L.E., 2007. Iprodione delays male

rat pubertal development, reduces serum testosterone levels, and decreases ex vivo

testicular testosterone production. Toxicology Letters 174 (1),7481.

Campos, M., Perruchon, C., Vasilieiadis, S., MenkissogluSpiroudi, U., Karpouzas, D.G., Diez,

M., 2015. Isolation and characterization of bacteria from acidic pristine soil environment

able to transform iprodione and 3, 5dichloraniline. International Biodeterioration &

Biodegradation 104, 201211.

12 https://mc06.manuscriptcentral.com/cjm-pubs Page 13 of 22 Canadian Journal of Microbiology

Campos, M., Karas, P.S., Perruchon, C., Papadopoulou, E.S., Christou, V.,

MenkissoglouSpiroudi, U., Diez, M.C., Karpouzas, D. G., 2017. Novel insights into the

metabolic pathway of iprodione by soil bacteria. Environmental Science and Pollution

Research 24(1): 152163.

Cao, L., Xu, J., Wu, G., Li, M., Jiang, J., He, J., Li, S., Hong, Q., 2013. Identification of two

combined genes responsible for dechlorination of 3,5,6trichloro2pyridinol (TCP) in

Cupriavidus pauculus P2. Journal of Hazardous Materials 260, 700706.

Carmona, M.A., Barreto, D.E., Reis, E.M., 2001. Effect of iprodione and its mixtures with

thiram and triticonazole to control Drechslera teres in barley seed. Fitopatologia

Brasileira 26 (2), 176179.

Chen, S., Hu, M., Liu, J., Zhong, G., Yang, L., RizwanulHaq, M., Han, H., 2011.

Biodegradation of betacypermethrinDraft and 3phenoxybenzoic acid by a novel

Ochrobactrum lupini DGS01. Journal of Hazardous Materials 187 (1), 433440.

Davidse, L.C., 1986. Benzimidazole fungicides: mechanism of action and biological impact.

Annual Review of Phytopathology 24 (1), 4365.

Derbalah, A.S.H., Nakatani, N., Sakugawa, H., 2003. Distribution, seasonal pattern, flux and

contamination source of pesticides and nonylphenol residues in Kurose River water,

HigashiHiroshima, Japan. Geochemical Journal 37 (2), 217232.

Grabke, A., FernándezOrtuño, D., Amiri, A., Li, X., Peres, N.A., Smith, P., Schnabel, G.,

2014. Characterization of iprodione resistance in Botrytis cinerea from strawberry and

blackberry. Phytopathology 104 (4), 396402.

Holt, J., Krieg, N., Sneath, P., Sneath, P.H., Staley, J.T., Williams, S.T., 1994. Bergey's manual

of determinative bacteriology. 9th Edn., Williams and Wilkins. Baltimore. 244254.

Lakshmi, V., Das, N., 2017. Biodegradation of atrazine by Cryptococcus laurentii isolated

from contaminated agricultural soil. Journal of Microbiology and Biotechnology

13 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 14 of 22

Research 2(3): 450457.

Lane, D.J., 1991. 16/23S rRNA sequencing. In: Stackerbrandt E, Goodfellow M (eds),

Nucleic acid techniques in bacterial systematics Wiley, New York. 115175.

Mercadier, C., Garcia, D., Vega, D., Bastide, J., Coste, C., 1996. Metabolism of iprodione in

adapted and nonadapted soils; Effect of soil inoculation with an iprodionedegrading

Arthrobacter strain. Soil Biology and Biochemistry 28(12), 17911796.

Mercadier, C., Vega, D., Bastide, J., 1997. Iprodione degradation by isolated soil

microorganisms. FEMS Microbiology Ecology 23 (3), 207215.

Miñambres, G.G., Conles, M.Y., Lucini, E.I., Verdenelli, R.A., Meriles, J.M., Zygadlo, J.A.,

2010. Application of thymol and iprodione to control garlic white rot ( Sclerotium

cepivorum ) and its effect on soil microbial communities. World Journal of Microbiology

and Biotechnology 26 (1), 161170.Draft

Morales, J., Manso, J.A., Cid, A., Mejuto, J.C., 2013. Stability study of iprodione in alkaline

media in the presence of humic acids. Chemosphere 92 (11), 15361541.

Munnecke, D.M., Hsieh, D.P.H., 1974. Microbial decontamination of parathion and

pnitrophenol in aqueous media. Applied Microbiology 28, 212217.

Omirou, M., Vryzas, Z., PapadopoulouMourkidou, E., Economou, A., 2009. Dissipation rates

of iprodione and thiacloprid during tomato production in greenhouse. Food Chemistry

116 (2), 499504.

Rajendran, P., Muthukrishnan, J., Gunasekaran, P., 2003. Microbes in heavy metal

remediation. Indian Journal of Experimental Biology 41 (9), 935944.

Saitou, N., Nei, M., 1987. The neighborjoining method: a new method for reconstructing

phylogenetic trees. Molecular Biology and Evolution 4 (4), 406425.

Sequinatto, L., Reichert, J.M., Santos, D.R., Reinert, D.J., Copetti, A.C.C., 2013. Occurrence

of agrochemicals in surface waters of shallow soils and steep slopes cropped to tobacco.

14 https://mc06.manuscriptcentral.com/cjm-pubs Page 15 of 22 Canadian Journal of Microbiology

Química Nova 36 (6), 768772.

Singh, B.K., Walker, A., 2006. Microbial degradation of organophosphorus compounds.

FEMS Microbiology Reviews 30, 428471.

Szeto, S.Y., Burlinson, N.E., Rahe, J.E., Oloffs, P.C., 1989. Kinetics of hydrolysis of the

dicarboximide fungicide vinclozolin. Journal of Agricultural and Food Chemistry 37 (2),

523529.

Verdenelli, R.A., Lamarque, A.L., Meriles, J.M., 2012. Shortterm effects of combined

iprodione and vermicompost applications on soil microbial community structure. Science

of the Total Environment 414, 210219.

Wang, X., Xu, G., Wang, F., Sun, H., Li, Y., 2012. Iprodione residues and dissipation rates in

tobacco leaves and soil. Bulletin of Environmental Contamination and Toxicology 89 (4),

877881. Draft

Wang, Y.S., Wen, C.Y., Chiu, T.C., Yen, J.H., 2004. Effect of fungicide iprodione on soil

bacterial community. Ecotoxicology and Environmental Safety 59 (1), 127132.

Washington, T., Tchounwou, P.B., 2004. Cytotoxicity and transcriptional activation of stress

genes in human liver carcinoma (HepG2) cells exposed to iprodione. International

Journal of Environmental Research and Public Health 1 (1), 1220.

Yadav, M., Srivastva, N., Singh, R.S., Upadhyay, S.N., Dubey, S.K, 2014. Biodegradation of

chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresource

Technology 165, 265269.

Yang, Z., Wu, G., Jin, W., Cao, L., Yan, X., Hong, Q., 2017. Isolation, identification and

characterization of an iprodionedegrading strain YJNG. Chinese Journal of Applied and

Environmental Biology 01, 19.

Zhang, C., Li, M., Xu, X., Liu, N., 2015. Effects of carbon nanotubes on atrazine

biodegradation by Arthrobacter sp. Journal of hazardous materials 287, 16.

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Figure Legends

Fig. 1 NeighborJoining tree showing the phylogenetic relationship between strain CQH1 and related species based on the 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are given at the nodes. The scale bar represents an evolutionary distance of 0.002. The accession numbers are in parentheses.

Fig. 2 Kinetics of degradation and the growth of strain CQH1with iprodione (A) or

3,5dichloroaniline (B) as the sole carbon source. The data are means±standard deviation for triplicate treatments

Fig.3 Effect of temperature on degradationDraft rate of iprodione in MSM containing 100 mg L1 after culturing for 64 h. The data are means ±standard deviation for triplicate.

Fig.4 Effect of pH on degradation rate of iprodione by strain CQH1 in MSM containing 100 mg L1 after culturing for 64 h. The data are means±standard deviation for triplicate.

Fig. 5 GCMass spectra of iprodione and its degradation products produced by

Microbacterium sp. strain CQH1.

Fig. 6 Proposed degradation pathway of iprodione by strain Microbacterium sp. CQH1.

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Figure 1

Fig. 1 Neighbor-Joining tree showing the phylogenetic relationship between strain CQH-1 and related species based on the 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are given at the nodes. The scale bar represents an evolutionary distance of 0.002. The accession numbers are in parentheses.

86 DSM 12966 T（JYIU01000006 ） 27 Microbacterium phyllosphaerae DSM 13468 T（AJ277840 ） 19 Microbacterium natoriense TNJL143-2T（AY566291 ） T 31 Microbacterium ginsengiterrae DCY37 （EU873314 ） 47 Microbacterium aerolatum V-73 T（AJ309929 ） 32 Microbacterium hydrocarbonoxydans DSM 16089 T（AJ698726 ） Microbacterium oleivorans DSM 16091 T（AJ698725 ） 3 27 Microbacterium keratanolyticum IFO 13309 T（AB004717 ） Draft T Microbacterium testaceum DSM 20166 （X77445 ） T 33 DMMZ 1710 （Y14699 ） 25 T 54 Microbacterium thalassium IFO 16060 （AB004713 ） 67 Microbacterium yannicii G72 T（FN547412 ） Microbacterium azadirachtae DSM 23848 T（JYIT01000023 ） 12 Shh49 T（EF623999 ） 99 Microbacterium esteraromaticum DSM 8609 T（Y17231 ） Microbacterium arabinogalactanolyticum IFO 14344 T（AB004715 ） 95 Strain CQH-1 (KX618653 ） Microbacterium paraoxydans CF36 T（AJ491806 ） T 66 Microbacterium luteolum IFO 15074 （AB004718 ） Microbacterium saperdae IFO 15038T（AB004719 ） 84 DSM 20578 T（Y17227 ） 40 T 93 Microbacterium maritypicum DSM 12512 （AJ853910 ） 46 Microbacterium liquefaciens DSM 20638 T（X77444 ）

0.002

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Figure 2

A

B Draft

Fig. 2 Kinetics of degradation and the growth of strain CQH-1with iprodione (A) or

3,5-dichloroaniline (B) as the sole carbon source. The data are means±standard deviation for triplicate treatments.

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Figure 3

100

80

60

40

Degradation rate (%) rate Degradation 20

0 20 25 30 35 40 Temperature ( ℃)

Draft Fig.3 Effect of temperature on degradation rate of iprodione in MSM containing 100

mg L -1 after culturing for 64 h. The data are means ±standard deviation for triplicate.

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Figure 4

100

80

60

40

Degradation rate (%) Degradation 20

0 4 5 6 7 8 DraftpH

Fig.4 Effect of pH on degradation rate of iprodione by strain CQH-1 in MSM containing 100 mg L-1 after culturing for 64 h. The data are means±standard deviation for triplicate.

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Figure 5

A

C B

A Draft B C Cl O Cl O Cl

H N N NH2 N N NH

Cl O O Cl O Cl

Fig. 5 GC-Mass spectra of iprodione and its degradation products produced by

Microbacterium sp. strain CQH-1.

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Figure 6

Cl O Cl O Cl Cl H N N NHCONHCH2COOH CO + H O N N NH NH2 2 2 Cl Cl O O Cl O Cl Iprodione N-(3,5-Dichlorophenyl)-2,4-dioxoimidazolidine 3,5-dichlorophenylurea acetic acid 3,5-Dichloroaniline

Fig. 6 Proposed degradation pathway of iprodione by strain Microbacterium sp.

CQH-1.

Draft

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